Effect of Nucleotide State on the Protofilament Conformation of Tubulin Octamers (original) (raw)
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Microtubules are essential parts of the cytoskeleton that are built by polymerization of tubulin heterodimers into a hollow tube. Regardless that their structures and functions have been comprehensively investigated in a modern soft matter, it is unclear how properties of tubulin heterodimer influence and promote the self-assembly. A detailed knowledge of such structural mechanisms would be helpful in drug design against neurodegenerative diseases, cancer, diabetes etc. In this work atomistic molecular dynamics simulations were used to investigate the fundamental dynamics of tubulin heterodimers in a sheet and a short microtubule utilizing well-equilibrated structures. The breathing motions of the tubulin heterodimers during assembly show that the movement at the lateral interface between heterodimers (wobbling) dominates in the lattice. The simulations of the protofilament curvature agrees well with recently published experimental data, showing curved protofilaments at polymerizati...
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Tubulin heterodimers are the building blocks of microtubules, a major component of the cytoskeleton, whose mechanical properties are fundamental for the life of the cell. We uncover the microscopic origins of the mechanical response in microtubules by probing features of the energy landscape of the tubulin monomers and tubulin heterodimer. To elucidate the structures of the unfolding pathways and reveal the multiple unfolding routes, we performed simulations of a self-organized polymer (SOP) model of tubulin. The SOP representation, which is a coarse-grained description of chains, allows us to perform force-induced simulations at loading rates and time scales that closely match those used in single-molecule experiments. We show that the forced unfolding of each monomer involves a bifurcation in the pathways to the stretched state. After the unfolding of the C-term domain, the unraveling continues either from the N-term domain or from the middle domain, depending on the monomer and the pathway. In contrast to the unfolding complexity of the monomers, the dimer unfolds according to only one route corresponding to the unraveling of the C-term domain and part of the middle domain of -tubulin. We find that this surprising behavior is due to the viscoelastic properties of the interface between the monomers. We map precise features of the complex energy landscape of tubulin by surveying the structures of the various metastable intermediates, which, in the dimer case, are characterized only by changes in the -tubulin monomer.
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2013
Microtubules are filaments within cells that play important roles in intracellular transport, cell division, and overall structural support. Microtubules are composed of tubulin dimers of α-and β-tubulin. The dimers polymerize at the (+) end of the microtubule to cause microtubule growth. β-tubulin must be bound to GTP for dimers to polymerize effectively. Microtubules will undergo catastrophic fraying and depolymerization at the (+) end when only GDP-bound tubulin is present. GTP-and GDP-bound dimers in solution seem to exist in a slightly bent state, while they must straighten to polymerize in the microtubule. While the effect of GTP on microtubule stability is clear, it is not so clear exactly how GTP influences the structural integrity of the microtubule, whether it causes conformational changes in the tubulin that prepare the tubulin for polymerization, or whether it facilitates the structural switch upon polymerization. Analysis of computer simulations of protofilaments in either the GTP-or GDP-bound state could elucidate how these nucleotide states affect the curvature of intra and inter dimer linkages and the force required for straightening. Our analysis indicated that nucleotide state did not influence the curvature or flexibility of solvated protofilaments in a way to facilitate polymerization. The GTP-bound protofilament had greater curvature by the end of simulation. Further analysis of protofilaments aligned to a microtubule image or with lateral contacts in place is recommended.
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The molecular basis of microtubule lattice instability derives from the hydrolysis of GTP to GDP in the lattice-bound state of Rβ-tubulin. While this has been appreciated for many years, there is ongoing debate over the molecular basis of this instability and the possible role of altered nucleotide occupancy in the induction of a conformational change in tubulin. The debate has organized around seemingly contradictory models. The allosteric model invokes nucleotide-dependent states of curvature in the free tubulin dimer, such that hydrolysis leads to pronounced bending and thus disruption of the lattice. The more recent lattice model describes a predominant role for the lattice in straightening free dimers that are curved regardless of their nucleotide state. In this model, lattice-bound GTP-tubulin provides the necessary force to straighten an incoming dimer. Interestingly, there is evidence for both models. The enduring nature of this debate stems from a lack of high-resolution data on the free dimer. In this study, we have prepared Rβ-tubulin samples at high dilution and characterized the nature of nucleotide-induced conformational stability using bottom-up hydrogen/deuterium exchange mass spectrometry (H/DX-MS) coupled with isothermal urea denaturation experiments. These experiments were accompanied by molecular dynamics simulations of the free dimer. We demonstrate an intermediate state unique to GDP-tubulin, suggestive of the curved colchicine-stabilized structure at the intradimer interface but show that intradimer flexibility is an important property of the free dimer regardless of nucleotide occupancy. Our results indicate that the assembly properties of the free dimer may be better described on the basis of this flexibility. A blended model of assembly emerges in which free-dimer allosteric effects retain importance, in an assembly process dominated by lattice-induced effects.
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Nature, 2006
Microtubules are highly dynamic protein polymers 1 that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro 2,3 and in vivo 4,5 , to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule endbinding proteins regulate microtubule dynamics in living cells 7-9 .
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European Biophysics Journal With Biophysics Letters, 2006
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